Lecture 5 SDS Polyacrylamide - PowerPoint Presentation

1. Lecture 5SDS Polyacrylamide GEByMSc. Elham FaisalMSc.Wafaa AjamAnalytical chemistry(practical) Ministry of higher education and scientific researchAL-Mustaqbal University college Department of medical physics

2. Polyacrylamide is a well-defined, stable, and rather inert gel with a pore size that can be easily varied.It is mechanically strong, easily handled, and transparent.Due to relatively small pore size, the eddy diffusion is low and hence contributes little to band broadening.However, due to the small pore size, polyacrylamide gels are less suitable for separation of very large molecules such as proteins with molecular mass . >300 000 (300 kDa).Polyacrylamide Gel

3. Understanding the mechanism of Polyacrylamide Gel Electrophoresis Techniques to separate proteins.

4. Remember the velocity of migration ( v ) of a protein (or any molecule) in an electric field depends on the electric field strength ( E ), the net charge on the protein ( z ), and the frictional coefficient ( f ).v = Ez/f …………………..(1)The electric force Ez driving the charged molecule toward the oppositely charged electrode is opposed by the viscous drag fv arising from friction between the moving molecule and the medium.The frictional coefficient f depends on both the mass and shape of the migrating molecule and the viscosity η of the medium. For a sphere of radius r , f = 6 η r ………………. (2)Proteins can be separated by gel electrophoresis

5. Electrophoretic separations are nearly always carried out in porous gels (or on solid supports such as paper) because the gel serves as a molecular sieve thatenhances separation (Figure 3).Figure 3 :Polyacrylamide gel electrophoresis .(A) Gel electrophoresis apparatus. Typically, several samples undergo electrophoresis on one flat polyacrylamide gel. Amicroliter pipette is used to place solutions of proteins in the wells of the slab. A cover is then placed over the gel chamber and voltage is applied. The negatively charged SDS (sodium dodecyl sulfate)–protein complexes migrate in the direction of the anode, at the bottom of the gel. (B) The sieving action of a porous polyacrylamide gel separates proteins according to size, with the smallest moving most rapidly.

6. Molecules that are small compared with the pores in the gel readily move through the gel, whereas molecules much larger than the pores are almost immobile.Intermediate-size molecules move through the gel with various degrees of facility.The electric field is applied such that proteins migrate from the negative to the positive electrodes, typically from top to bottom.Electrophoresis is performed in a thin, vertical slab of polyacrylamide gel.

7. Polyacrylamide gels are choice supporting media for electrophoresis because they are chemically inert and readily formed by the polymerization of acrylamide with a small amount of the cross-linking agent methylenebisacrylamide to make a three dimensional mesh (Figure 4).Electrophoresis is distinct from gel filtration in that, because of the electric field, all of the molecules, regardless of size, are forced to move through the same matrix

8. Figure 4: Formation of a polyacrylamide gel. A three-dimensional mesh is formed by copolymerizing activated monomer (blue) and cross-linker (red).

9. Proteins can be separated largely on the basis of mass by electrophoresis in a polyacrylamide gel under denaturing conditions.The mixture of proteins is first dissolved in a solution of sodium dodecylsulfate (SDS), an anionic detergent that disrupts nearly all non-covalent interactions in native proteins.B -Mercaptoethanol (2-thioethanol) or dithiothreitol is added to reduce disulfide bonds. Anions of SDS bind to main chains at a ratio of about one SDS anion for every two amino acid residues.The negative charge acquired on binding SDS is usually much greater than the charge on the native protein; the contribution of the protein to the total charge of the SDS–protein complex is thus rendered insignificant.

10. As a result, this complex of SDS with a denatured protein has a large net negative charge that is roughly proportional to the mass of the protein.The SDS–protein complexes are then subjected to electrophoresis.When the electrophoresis is complete, the proteins in the gel can be visualized by staining them with silver nitrate or a dye such as Coomassie blue, which reveals a series of bands (Figure 5).Figure (5):Staining of proteins after electrophoresis . Mixtures of proteins from cellular extracts subjected to electrophoresis on an SDS polyacrylamide gel can be visualized by staining with Coomassie blue. The first lane contains a mixture of proteins of known molecular weights, which can be used to estimate the sizes of the bands in the samples.

11. Radioactive labels, if they have been incorporated into proteins, can be detected by placing a sheet of x-ray film over the gel, a procedure called autoradiography. Small proteins move rapidly through the gel, whereas large proteins stay at the top, near the point of application of the mixture.The mobility of most polypeptide chains under these conditions is linearly proportional to the logarithm of their mass figure (6).Figure (6):Electrophoresis can determine mass. The electrophoretic mobility of many proteins in SDS–polyacrylamide gels is inversely proportional to the logarithm of their mass.

12. Some carbohydrate-rich proteins and membrane proteins do not obey this empirical relation, however. SDS–polyacrylamide gel electrophoresis (often referred to as SDS-PAGE) is rapid, sensitive, and capable of a high degree of resolution. As little as 0.1 µg (~2 pmol) of a protein gives a distinct band when stained with Coomassie blue, and even less (~0.02 µg) can detected with a silver stain.Proteins that differ in mass by about 2% (e.g., 50 and 51 kDa, arising from adifference of about10 amino acids) can usually be distinguished with SDS-PAGE.We can examine the efficacy of our purification scheme by analyzing a part of each fraction by electrophoresis.The initial fractions will display dozens to hundreds of proteins. As the purification progresses, the number of bands will diminish, and the prominence of one of the bands should increase. This band should correspond to the protein of interest.

13. How can we discover the identity of a protein that is showing such responses? Although many proteins are displayed on a two-dimensional gel, they are not identified.It is now possible to identify proteins by coupling two-dimensional gel electrophoresis with mass spectrometric techniques .

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